SnapShot: Origins of DNA Replication Rachel L. Creager, Yulong Li, and David M. MacAlpine Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC 27710, USA E. coli Number of origins
S. cerevisiae
1
Genome size 4.6 Mb
Human
Number of origins ~350
Number of origins
Genome size
Genome size
12.5 Mb
40,000–80,000
3.3 Gb NFR
NFR
oriC
IHF DUE
ARS1
G-quadruplex
FIS
R1
R2
R3
R4
ACS
B1
G3-5N1-7G3-5N1-7G3-5N1-7G3-5
B2
DnaA ORC
ORC CpG
FIS Cdt1 DnaB
ORC
ORC CpG
Cdc6 MCM DnaC
IHF
ORC
ORC
CpG
Histone Ptm
418
enzyme
function
organism
H4K20me1
PR-Set7
Promotes pre-RC assembly
Human, mouse
H4K20me2
Suv4-20h1/2
ORC recruitment via ORC1 BAH domain
Metazoa
H3K4me2
COMPASS complex
Origin activation
Yeast
H3K27me
ATXR5, ATRX6
Represses re-replication of heterochromatic origins
Arabidopsis
H3K36me
Set2
Regulates Cdc45 association with origins
Yeast
H3K79me2
DOT1L
Enriched at origins, loss of DOT1L leads to re-replication
Human
H3K4me3 demethylation
KDM5C/JARID1C
Promotes early-origin activation
Human
H4Ac
Hbo1
Promotes pre-RC assembly
Human, xenopus, fly
Bulk H3 and H4 acetylation
Multiple HATs
Regulates developmentally programmed origin of the β-globin locus
Human
H4K5Ac/H4K12Ac
Hat1p/Hat2p
Hat1/Hat2 interact with and enhance the function of ORC
Yeast
H4K16Ac
MOF
Promotes male-specific early-origin activation on the X chromosome
Fly
H3,H4 deacetylation
Rpd3
Delays late-origin activation, developmental transition in origin specificity
Yeast, fly
H3,H4 deacetylation
Sir2p
Inhibits pre-RC assembly at a subset of origins
Yeast
H4K5Ac deacetylation
Sum1/Rfm1/Hst1
Enhances efficiency of replication initiation at a subset of origins
Yeast
H2B Ub
Bre1
Enriched at origins, impacts fork elongation
Yeast
Cell 161, April 9, 2015 ©2015 Elsevier Inc. DOI http://dx.doi.org/10.1016/j.cell.2015.03.043
See online version for legend and references.
SnapShot: Origins of DNA Replication Rachel L. Creager, Yulong Li, and David M. MacAlpine Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC 27710, USA
More than 50 years ago, Jacob et al. (1964) proposed an elegant model for the regulation of DNA replication in bacteria. In the replicon model, the fundamental unit of DNA replication, the replicon, would be governed by a cis-acting replicator sequence and a trans-activating initiator factor. Despite the increased size and complexity of eukaryotic genomes, eukaryotic DNA replication continues to be guided by the fundamental principles and concepts established in the replicon model. Eukaryotic origins of replication (replicators) are defined by cis-acting sequences or structural DNA elements that are recognized by functionally conserved trans-acting initiator factors (ORC, Cdc6, Cdt1, and Mcm2-7). Prokaryotic Replication Origins E. coli has a single circular chromosome that is ~4.6 megabases in length, containing a single origin of replication (oriC). The two major cis-acting features of the ~250 bp oriC are an AT-rich DNA unwinding element (DUE) and multiple 9 bp DnaA-binding motifs (Skarstad and Katayama, 2013). DnaA is a AAA+ ATPase that recognizes both high- and low-affinity binding sites throughout oriC. High-affinity DnaA binding sites (R1–R4) are occupied throughout most of the cell cycle; however, low-affinity binding sites become occupied only at replication initiation. Two accessory proteins, Fis and IHF, repress or stimulate initiation of DNA replication, respectively, by altering the conformation of DNA surrounding oriC. Binding of a full complement of DnaA molecules (10–20) leads to DNA unwinding at DUE. This unwinding stimulates DnaC, a AAA+ ATPase helicase loader, to assemble two homohexamers of the DnaB helicase complex on the newly melted DNA, forming the pre-replicative complex (pre-RC) (Costa et al., 2013). Eukaryotic DNA Replication Origins A consequence of the large size of eukaryotic genomes is that multiple origins of replication must be utilized in parallel to facilitate the complete duplication of the genome within the confines of S phase (Gilbert, 2004). There are ~350 origins of replication distributed throughout the S. cerevisiae genome. In contrast, there are an estimated 40,000– 80,000 origins distributed throughout the much larger human genome. As in bacteria, both cis- and trans-acting factors define start sites of eukaryotic DNA replication. Eukaryotic cis-acting replicator elements were first identified in the model organism S. cerevisiae as autonomous replicating sequence (ARS) elements of ~200 bp. Each ARS element contains a conserved ARS consensus sequence (ACS), which, at its core, is an 11 bp T-rich motif that is necessary but not sufficient for origin function. In addition to the ACS, there are several other poorly defined B sequence elements that contribute to helicase loading and DNA unwinding. In contrast, conserved replicator sequences that direct origin selection in higher eukaryotes have remained elusive. Recently, low-complexity GC-rich sequences that are able to form G-quadruplexes have been identified as potential replicator elements, suggesting that DNA secondary structure may play a role in origin licensing in higher eukaryotes (Cayrou et al., 2012). There is remarkable functional conservation between prokaryotic and eukaryotic trans-acting initiator factors. Analogous to DnaA, the origin recognition complex (ORC) recognizes and binds to replicator origin sequences throughout the majority of the cell cycle. ORC is composed of six subunits, five of which are AAA+ ATPases. In G1 of the cell cycle, another initiation factor and AAA+ ATPase, Cdc6, associates with ORC and coordinates the loading of a double hexamer of the minichromosome maintenance (Mcm2–7) helicase complex via an interaction with Cdt1 to form the pre-RC (Bell and Kaguni, 2013). Formation of the pre-RC “licenses” the origin for potential activation in the subsequent S phase. Eukaryotic cis-acting replicator elements are necessary but not sufficient for origin activity, as there are many more ACS motif matches and potential G-quadruplexes than utilized origins of replication. Epigenetic features, including promoters, CpG islands, nucleosome organization, and post-translational modification of histones, also impact the selection and activation of eukaryotic replication origins (Ding and MacAlpine, 2011). A nucleosome-free region (NFR) with well-positioned flanking nucleosomes is a conserved feature of eukaryotic replicator sequences, and maintenance of the nucleosome free region at the origin is critical for function. Eukaryotic origins are typically associated with intergenic sequences. In higher eukaryotes, origins and ORC binding sites are frequently found in the NFR associated with transcription start sites and CpG islands, whereas in S. cerevisiae, ORC is excluded from transcription start sites. A diverse array of histone PTMs are correlated with ORC binding and origin function; however, only a handful of epigenetic marks have been mechanistically linked to specific steps in origin selection and activation (Dorn and Cook, 2011; Méchali et al., 2013). The methylation state of histone H4 lysine 20 in metazoans has been linked to ORC binding and pre-RC assembly. Dimethylation of H4K20 by the methyltransferase Suv4-20h1/2 is recognized by the bromo-adjacent homology (BAH) domain of ORC1. In addition, monomethylation of H4K20 by the cell-cycle-regulated methyltransferase, PR-Set7, promotes pre-RC assembly. The histone acetyltransferase (HAT) Hbo1 interacts with ORC, Cdt1, and Mcm2 and is required for efficient pre-RC assembly. Presumably, Hbo1 acetylates origin-proximal nucleosomes on histone H4 to promote pre-RC assembly; however, it remains possible that the target of Hbo1 is not histones but instead specific pre-RC components. Finally, although numerous epigenetic modifications and chromatin states have been correlated with origin function, it is important to stress that correlation does not equal causation. The S. cerevisiae histone deacetylase Rpd3 represses global origin activation not by deacetylating histone H3 in the vicinity of replication origins but rather by regulating silencing of the origin-rich multicopy rDNA locus, which serves as a sink for sequestering key replication initiation factors (Yoshida et al., 2014). Acknowledgments We thank members of the MacAlpine group for ideas and suggestions. This work was supported by NIH grants R01 GM104097 (D.M.M.) and T-32 CA059365 (R.L.C.). References Bell, S.P., and Kaguni, J.M. (2013). Cold Spring Harb. Perspect. Biol. 5, a010124. Cayrou, C., Coulombe, P., Puy, A., Rialle, S., Kaplan, N., Segal, E., and Méchali, M. (2012). Cell Cycle 11, 658–667. Costa, A., Hood, I.V., and Berger, J.M. (2013). Annu. Rev. Biochem. 82, 25–54. Ding, Q., and MacAlpine, D.M. (2011). Crit. Rev. Biochem. Mol. Biol. 46, 165–179. Dorn, E.S., and Cook, J.G. (2011). Epigenetics 6, 552–559. Gilbert, D.M. (2004). Nat. Rev. Mol. Cell Biol. 5, 848–855. Jacob, F., Brenner, S., and Cuzin, F. (1964). Cold Spring Harb. Symp. Quant. Biol. 28, 329. Méchali, M., Yoshida, K., Coulombe, P., and Pasero, P. (2013). Curr. Opin. Genet. Dev. 23, 124–131. Skarstad, K., and Katayama, T. (2013). Cold Spring Harb. Perspect. Biol. 5, a012922. Yoshida, K., Bacal, J., Desmarais, D., Padioleau, I., Tsaponina, O., Chabes, A., Pantesco, V., Dubois, E., Parrinello, H., Skrzypczak, M., et al. (2014). Mol. Cell 54, 691–697.
418.e1 Cell 161, April 9, 2015 ©2015 Elsevier Inc. DOI http://dx.doi.org/10.1016/j.cell.2015.03.043
S. cerevisiae
1
Genome size 4.6 Mb
Human
Number of origins ~350
Number of origins
Genome size
Genome size
12.5 Mb
40,000–80,000
3.3 Gb NFR
NFR
oriC
IHF DUE
ARS1
G-quadruplex
FIS
R1
R2
R3
R4
ACS
B1
G3-5N1-7G3-5N1-7G3-5N1-7G3-5
B2
DnaA ORC
ORC CpG
FIS Cdt1 DnaB
ORC
ORC CpG
Cdc6 MCM DnaC
IHF
ORC
ORC
CpG
Histone Ptm
418
enzyme
function
organism
H4K20me1
PR-Set7
Promotes pre-RC assembly
Human, mouse
H4K20me2
Suv4-20h1/2
ORC recruitment via ORC1 BAH domain
Metazoa
H3K4me2
COMPASS complex
Origin activation
Yeast
H3K27me
ATXR5, ATRX6
Represses re-replication of heterochromatic origins
Arabidopsis
H3K36me
Set2
Regulates Cdc45 association with origins
Yeast
H3K79me2
DOT1L
Enriched at origins, loss of DOT1L leads to re-replication
Human
H3K4me3 demethylation
KDM5C/JARID1C
Promotes early-origin activation
Human
H4Ac
Hbo1
Promotes pre-RC assembly
Human, xenopus, fly
Bulk H3 and H4 acetylation
Multiple HATs
Regulates developmentally programmed origin of the β-globin locus
Human
H4K5Ac/H4K12Ac
Hat1p/Hat2p
Hat1/Hat2 interact with and enhance the function of ORC
Yeast
H4K16Ac
MOF
Promotes male-specific early-origin activation on the X chromosome
Fly
H3,H4 deacetylation
Rpd3
Delays late-origin activation, developmental transition in origin specificity
Yeast, fly
H3,H4 deacetylation
Sir2p
Inhibits pre-RC assembly at a subset of origins
Yeast
H4K5Ac deacetylation
Sum1/Rfm1/Hst1
Enhances efficiency of replication initiation at a subset of origins
Yeast
H2B Ub
Bre1
Enriched at origins, impacts fork elongation
Yeast
Cell 161, April 9, 2015 ©2015 Elsevier Inc. DOI http://dx.doi.org/10.1016/j.cell.2015.03.043
See online version for legend and references.
SnapShot: Origins of DNA Replication Rachel L. Creager, Yulong Li, and David M. MacAlpine Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC 27710, USA
More than 50 years ago, Jacob et al. (1964) proposed an elegant model for the regulation of DNA replication in bacteria. In the replicon model, the fundamental unit of DNA replication, the replicon, would be governed by a cis-acting replicator sequence and a trans-activating initiator factor. Despite the increased size and complexity of eukaryotic genomes, eukaryotic DNA replication continues to be guided by the fundamental principles and concepts established in the replicon model. Eukaryotic origins of replication (replicators) are defined by cis-acting sequences or structural DNA elements that are recognized by functionally conserved trans-acting initiator factors (ORC, Cdc6, Cdt1, and Mcm2-7). Prokaryotic Replication Origins E. coli has a single circular chromosome that is ~4.6 megabases in length, containing a single origin of replication (oriC). The two major cis-acting features of the ~250 bp oriC are an AT-rich DNA unwinding element (DUE) and multiple 9 bp DnaA-binding motifs (Skarstad and Katayama, 2013). DnaA is a AAA+ ATPase that recognizes both high- and low-affinity binding sites throughout oriC. High-affinity DnaA binding sites (R1–R4) are occupied throughout most of the cell cycle; however, low-affinity binding sites become occupied only at replication initiation. Two accessory proteins, Fis and IHF, repress or stimulate initiation of DNA replication, respectively, by altering the conformation of DNA surrounding oriC. Binding of a full complement of DnaA molecules (10–20) leads to DNA unwinding at DUE. This unwinding stimulates DnaC, a AAA+ ATPase helicase loader, to assemble two homohexamers of the DnaB helicase complex on the newly melted DNA, forming the pre-replicative complex (pre-RC) (Costa et al., 2013). Eukaryotic DNA Replication Origins A consequence of the large size of eukaryotic genomes is that multiple origins of replication must be utilized in parallel to facilitate the complete duplication of the genome within the confines of S phase (Gilbert, 2004). There are ~350 origins of replication distributed throughout the S. cerevisiae genome. In contrast, there are an estimated 40,000– 80,000 origins distributed throughout the much larger human genome. As in bacteria, both cis- and trans-acting factors define start sites of eukaryotic DNA replication. Eukaryotic cis-acting replicator elements were first identified in the model organism S. cerevisiae as autonomous replicating sequence (ARS) elements of ~200 bp. Each ARS element contains a conserved ARS consensus sequence (ACS), which, at its core, is an 11 bp T-rich motif that is necessary but not sufficient for origin function. In addition to the ACS, there are several other poorly defined B sequence elements that contribute to helicase loading and DNA unwinding. In contrast, conserved replicator sequences that direct origin selection in higher eukaryotes have remained elusive. Recently, low-complexity GC-rich sequences that are able to form G-quadruplexes have been identified as potential replicator elements, suggesting that DNA secondary structure may play a role in origin licensing in higher eukaryotes (Cayrou et al., 2012). There is remarkable functional conservation between prokaryotic and eukaryotic trans-acting initiator factors. Analogous to DnaA, the origin recognition complex (ORC) recognizes and binds to replicator origin sequences throughout the majority of the cell cycle. ORC is composed of six subunits, five of which are AAA+ ATPases. In G1 of the cell cycle, another initiation factor and AAA+ ATPase, Cdc6, associates with ORC and coordinates the loading of a double hexamer of the minichromosome maintenance (Mcm2–7) helicase complex via an interaction with Cdt1 to form the pre-RC (Bell and Kaguni, 2013). Formation of the pre-RC “licenses” the origin for potential activation in the subsequent S phase. Eukaryotic cis-acting replicator elements are necessary but not sufficient for origin activity, as there are many more ACS motif matches and potential G-quadruplexes than utilized origins of replication. Epigenetic features, including promoters, CpG islands, nucleosome organization, and post-translational modification of histones, also impact the selection and activation of eukaryotic replication origins (Ding and MacAlpine, 2011). A nucleosome-free region (NFR) with well-positioned flanking nucleosomes is a conserved feature of eukaryotic replicator sequences, and maintenance of the nucleosome free region at the origin is critical for function. Eukaryotic origins are typically associated with intergenic sequences. In higher eukaryotes, origins and ORC binding sites are frequently found in the NFR associated with transcription start sites and CpG islands, whereas in S. cerevisiae, ORC is excluded from transcription start sites. A diverse array of histone PTMs are correlated with ORC binding and origin function; however, only a handful of epigenetic marks have been mechanistically linked to specific steps in origin selection and activation (Dorn and Cook, 2011; Méchali et al., 2013). The methylation state of histone H4 lysine 20 in metazoans has been linked to ORC binding and pre-RC assembly. Dimethylation of H4K20 by the methyltransferase Suv4-20h1/2 is recognized by the bromo-adjacent homology (BAH) domain of ORC1. In addition, monomethylation of H4K20 by the cell-cycle-regulated methyltransferase, PR-Set7, promotes pre-RC assembly. The histone acetyltransferase (HAT) Hbo1 interacts with ORC, Cdt1, and Mcm2 and is required for efficient pre-RC assembly. Presumably, Hbo1 acetylates origin-proximal nucleosomes on histone H4 to promote pre-RC assembly; however, it remains possible that the target of Hbo1 is not histones but instead specific pre-RC components. Finally, although numerous epigenetic modifications and chromatin states have been correlated with origin function, it is important to stress that correlation does not equal causation. The S. cerevisiae histone deacetylase Rpd3 represses global origin activation not by deacetylating histone H3 in the vicinity of replication origins but rather by regulating silencing of the origin-rich multicopy rDNA locus, which serves as a sink for sequestering key replication initiation factors (Yoshida et al., 2014). Acknowledgments We thank members of the MacAlpine group for ideas and suggestions. This work was supported by NIH grants R01 GM104097 (D.M.M.) and T-32 CA059365 (R.L.C.). References Bell, S.P., and Kaguni, J.M. (2013). Cold Spring Harb. Perspect. Biol. 5, a010124. Cayrou, C., Coulombe, P., Puy, A., Rialle, S., Kaplan, N., Segal, E., and Méchali, M. (2012). Cell Cycle 11, 658–667. Costa, A., Hood, I.V., and Berger, J.M. (2013). Annu. Rev. Biochem. 82, 25–54. Ding, Q., and MacAlpine, D.M. (2011). Crit. Rev. Biochem. Mol. Biol. 46, 165–179. Dorn, E.S., and Cook, J.G. (2011). Epigenetics 6, 552–559. Gilbert, D.M. (2004). Nat. Rev. Mol. Cell Biol. 5, 848–855. Jacob, F., Brenner, S., and Cuzin, F. (1964). Cold Spring Harb. Symp. Quant. Biol. 28, 329. Méchali, M., Yoshida, K., Coulombe, P., and Pasero, P. (2013). Curr. Opin. Genet. Dev. 23, 124–131. Skarstad, K., and Katayama, T. (2013). Cold Spring Harb. Perspect. Biol. 5, a012922. Yoshida, K., Bacal, J., Desmarais, D., Padioleau, I., Tsaponina, O., Chabes, A., Pantesco, V., Dubois, E., Parrinello, H., Skrzypczak, M., et al. (2014). Mol. Cell 54, 691–697.
418.e1 Cell 161, April 9, 2015 ©2015 Elsevier Inc. DOI http://dx.doi.org/10.1016/j.cell.2015.03.043
